The continued goal of this Initiative is to determine the high resolution structures of two major[unreadable] migration organelles, adhesions and protrusions. Our approach is to combine structural methods that range[unreadable] from correlative light and high resolution electron microscopy to X-ray and NMR. As part of this effort, we are[unreadable] developing a new imaging concept that allows us to capture by light microscopy the spatial-temporal aspects of[unreadable] migration in living cells and to visualize the same structures using high resolution electron microscopy - 3D[unreadable] correlative light and electron microscopy ("top down approaches"). High resolution structures of individual[unreadable] proteins and their detailed interactions with binding partners obtained by NMR, X-ray or tomography ("bottom[unreadable] up approaches") can then be computationally docked into the three-dimensional maps of the complexes. In the[unreadable] first phase of the Consortium, this paradigm was applied to the structure of the actin network in protrusions,[unreadable] providing 3D structures of Arp2/3 mediated actin branches and the structural basis for Arp2/3 activation. We[unreadable] also determined the ultrastructure of the lamellipodia of living cells using our new 3D correlative EM and[unreadable] speckle microscopy and determined the vinculin binding site on a-actinin in the presence of integrin. A unified[unreadable] framework for segmentation and analysis of 3-D data was developed to enable the identification of assemblies[unreadable] in 3-D structures.